PROSTVAC-VF comprises two recombinant viral vectors, each encoding transgenes for prostate-specific antigen (PSA), and three immune costimulatory molecules (B7.1, ICAM-1, and LFA-3, designated TRICOM). Vaccinia-based vector was used for priming followed by six planned fowlpox-based vector boosts. PROSTVAC-VF treatment was evaluated for safety and for prolongation of progression-free survival (PFS) and overall survival (OS) in a randomized, controlled, and blinded phase II study. In total, 125 patients were randomly assigned in a multicenter trial of vaccination series. Eligible patients had minimally symptomatic castration-resistant metastatic prostate cancer (mCRPC). Patients were allocated (2:1) to PROSTVAC-VF plus granulocyte-macrophage colony-stimulating factor or to control empty vectors plus saline injections. Eighty-two patients received PROSTVAC-VF and 40 received control vectors. Patient characteristics were similar in both groups. The primary end point was PFS, which was similar in the two groups (P = .6). However, at 3 years post study, PROSTVAC-VF patients had a better OS with 25 (30%) of 82 alive versus 7 (17%) of 40 controls, longer median survival by 8.5 months (25.1 v 16.6 months for controls), an estimated hazard ratio of 0.56, and stratified log-rank P = .0061. PROSTVAC-VF immunotherapy was well tolerated and associated with a 44% reduction in the death rate and an 8.5-month improvement in median OS in men with mCRPC. These provocative data provide preliminary evidence of clinically meaningful benefit but need to be confirmed in a larger phase III study. A concurrent multicenter, randomized Phase II trial employing PROSTVAC-VF provided further evidence of enhanced median OS (p = 0.0061) in patients with mCRPC. Thirty-two patients were vaccinated once with recombinant vaccinia containing the transgenes for PSA and three costimulatory molecules. Patients received boosters with recombinant fowlpox containing the same four transgenes. Twelve of 32 patients showed declines in serum PSA post-vaccination and 2/12 showed decreases in index lesions. Median OS was 26.6 months (predicted median OS by the Halabi nomogram was 17.4 months). Patients with greater PSA-specific T-cell responses showed a trend (p = 0.055) toward enhanced survival. In solid tumors such as prostate cancer, novel paradigms are needed to assess therapeutic efficacy. We utilized a method estimating tumor growth and regression rate constants from serial PSA measurements, and assessed its potential in patients with metastatic castration resistant prostate carcinoma (mCRPC). Patients were enrolled in five phase II studies, including an experimental vaccine trial, representing the evolution of therapy in mCRPC. Growth rate constants correlated with survival, except in patients receiving vaccine-based therapy where the evidence demonstrates prolonged survival presumably due to immunity developing subsequent to vaccine administration. The PSA-TRICOM vaccine appears to have provided marked benefit not apparent during vaccination, but consistent with subsequent development of a beneficial immune response. A recently completed study in mCRPC demonstrated that when ipilimumab (anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) monoclonal antibody) was combined with PSA-TRICOM (PROSTVAC) at escalating doses, the median survival was 34 months, which compares favorably to previous vaccine trials in mCRPC that resulted in median survivals of approximately 26 months. These and other data support the rationale for randomized studies employing immune checkpoint inhibitors in combination with TRICOM-based vaccines. Two of the most widely studied human tumor-associated antigens (TAAs) are CEA and mucin-1 (MUC-1). CEA is overexpressed in a wide range of human carcinomas, including gastrointestinal, breast, lung, pancreatic, medullary thyroid, ovarian, and prostate. MUC-1 is a tumor-associated mucin, which is overexpressed and hypoglycosylated in all human carcinomas as well as in acute myeloid leukemia (AML) and multiple myeloma. Studies have demonstrated that the C-terminus of MUC-1 functions as an oncogene. A study was conducted to obtain preliminary evidence of clinical response in metastatic breast and ovarian cancer patients with PANVAC (rV,rF-CEA-MUC1-TRICOM). A randomized multicenter study has been initiated evaluating docetaxel vs docetaxel plus PANVAC vaccine (rV-, rF-CEA-MUC1-TRICOM) in patients (n=48) with metastatic breast carcinoma. Preliminary findings to date indicate a substantial increase in time to progression in the combination arm vs the docetaxel alone arm. A Phase I study of intraprostatic vaccine administration in men with locally recurrent or progressive prostate cancer has been completed. The primary end point of this study was to determine the safety and feasibility of intraprostatic administration of PSA-TRICOM vaccine in patients with locally recurrent or progressive prostate cancer. This trial was a standard 3+ 3 dose escalation with 6 patients each in cohorts 4 and 5 to gather more immunologic data. Nineteen of 21 patients enrolled had locally recurrent prostate cancer after definitive radiation therapy, and 2 had no local therapy. All cohorts received initial subcutaneous vaccination with recombinant vaccinia (rV)-PSA-TRICOM and intraprostatic booster vaccinations with recombinant fowlpox (rF)-PSA-TRICOM. There were no dose-limiting toxicities. Overall, 19 of 21 patients on trial had stable (10) or improved (9) PSA values. There was a marked increase in CD4+ (p = 0.0002) and CD8+ (p = 0.0002) tumor infiltrates in post- versus pre-treatment tumor biopsies. Four of 9 patients evaluated had peripheral immune responses to PSA or NGEP. Intraprostatic administration of PSA-TRICOM is safe and feasible and can generate a significant immunologic response. Improved serum PSA kinetics and intense post-vaccination inflammatory infiltrates were seen in the majority of patients. Methods to predict clinical outcomes of therapeutic cancer vaccines will advance the field and provide new insights into critical determinants of in vivo efficacy. A retrospective study included 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus-based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. A glycan microarray was used to profile prevaccination antiglycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. The screen for predictive biomarkers identified antiglycan antibodies that consistently stratified subjects into groups with different Kaplan-Meier survival estimates. Prevaccination antibody levels to blood group A trisaccharide (BG-Atri) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with abundant anti-BG-Atri immunoglobulinM(IgM). This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, PSA.